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Abstract:

A kit is disclosed that includes a first component comprising alginate,
wherein the first component is comprised in a first sterile vial, and a
second component comprising cells comprising keratinocytes or
fibroblasts, or mixtures thereof, that secrete one or more biologically
active molecules selected from the group consisting of GM-CSF, VEGF, KGF,
bFGF, TGFβ, angiopoietin, EGF, IL-Iβ, IL-6, IL-8, TGFα,
and TNFα, wherein the cells are allogeneic and mitotically
inactive, a buffered solution, and human serum albumin or a
cryoprotectant, wherein the second component is comprised in a second
sterile vial.

Claims:

1-42. (canceled)

43. A cell preparation comprising mitotically inactive allogeneic cells
that are capable of secreting one or more biologically active molecules
selected from the group consisting of GM-CSF, VEGF, KGF, bFGF, TGFβ,
angiopoietin, EGF, IL-ID, IL-6, IL-8, TGFα, and TNFα, wherein
the cells are keratinocytes or fibroblasts, or mixtures thereof.

44. The cell preparation of claim 43, wherein the cells are mitotically
inactivated by irradiation.

45. The cell preparation of claim 43, wherein the cells are
differentiated.

46. The cell preparation of claim 43, wherein the concentration of cells
are in the range of about 1.times.10.sup.3 cells/μl to about
50.times.10.sup.3 cells/μl.

47. The cell preparation of claim 43, wherein the cells are a mixture of
keratinocytes and fibroblasts and the ratio of keratinocytes to
fibroblasts is 1:9.

48. The cell preparation of claim 43, wherein the cell preparation is
comprised within a biologically acceptable carrier.

52. The cell preparation of claim 43, wherein the cell preparation is
comprised in a wound dressing.

53. The cell preparation of claim 43, wherein the cell preparation is
comprised in an extracellular matrix or matrix material.

54. The cell preparation of claim 43, wherein the cell preparation is in
the form of a paste.

55. The cell preparation of claim 43, wherein the cell preparation is in
the form of a spray.

56. The cell preparation of claim 43, further comprising a
cryoprotectant.

57. The cell preparation of claim 56, wherein the cell preparation is
cryopreserved.

58. The cell preparation of claim 43, wherein the cell preparation is
comprised in a container.

59. The cell preparation of claim 43, wherein the cell preparation is
comprised in a sterile vial.

Description:

FIELD OF THE INVENTION

[0001] The present invention relates generally to tissue regeneration,
e.g., the treatment of wounds using growth factor-, cytokine-, or
angiogenic factor-secreting cells admixed with a biological or synthetic
extracellular matrix and/or attached or applied to a wound dressing or
solid nondegradable support matrix.

BACKGROUND OF THE INVENTION

[0002] Wounds (i.e., lacerations or openings) in mammalian tissue can
result in tissue disruption and coagulation of the microvasculature at
the wound face. Repair of such tissue represents an orderly, controlled
cellular response to injury. All soft tissue wounds, regardless of size,
heal in a similar manner. The mechanisms of tissue growth and repair are
biologic systems wherein cellular proliferation and angiogenesis occur in
the presence of an oxygen gradient. The sequential morphological and
structural changes, which occur during tissue repair have been
characterized in great detail and have, in some instances, been
quantified. See Hunt, T. K., et al., "Coagulation and macrophage
stimulation of angiogenesis and wound healing," in The surgical wound,
pp. 1-18, ed. F. Dineen & G. Hildrick-Smith (Lea & Febiger, Philadelphia:
1981).

[0003] Tissue regeneration in various organs, such as, e.g., the skin or
the heart depends on connective tissue restoring blood supply and
enabling residual organ-specific cells such as keratinocytes or muscle
cells to reestablish organ integrity. Thus, a relevant function of the
mesenchymal cells, i.e., the fibroblasts or, in addition, the endothelial
cells of vasculature, is secretion of factors enhancing the healing
process, e.g., factors promoting formation of new blood vessels
(angiogenesis) or factors promoting re-epithelialization by proliferating
and migrating keratinocytes.

[0004] The cellular morphology of a wound consists of three distinct
zones. The central avascular wound space is oxygen deficient, acidotic
and hypercarbic, and has high lactate levels. Adjacent to the wound space
is a gradient zone of local ischemia, which is populated by dividing
fibroblasts. Behind the leading zone is an area of active collagen
synthesis characterized by mature fibroblasts and numerous newly formed
capillaries (i.e., neovascularization). While new blood vessel growth
(angiogenesis) is necessary for the healing of wound tissue, angiogenic
agents generally are unable to fulfill the long-felt need of providing
the additional biosynthetic effects of tissue repair. Despite the need
for more rapid healing of wounds (i.e., severe burns, surgical incisions,
lacerations and other trauma), to date there has been only limited
success in accelerating wound healing with pharmacological agents.

[0005] The primary goal in the treatment of wounds is to achieve wound
closure. Open cutaneous wounds represent one major category of wounds.
This category includes acute surgical and traumatic, e.g., chronic
ulcers, burn wounds, as well as chronic wounds such as neuropathic
ulcers, pressure sores, arterial and venous (stasis) or mixed
arterio-venous ulcers, and diabetic ulcers. Open cutaneous wounds
routinely heal by a process comprising six major components: i)
inflammation, ii) fibroblast proliferation, iii) blood vessel
proliferation, iv) connective tissue synthesis, v) epithelialization, and
yl) wound contraction. Wound healing is impaired when these components,
either individually or as a whole, do not function properly. Numerous
factors can affect wound healing, including malnutrition, infection,
pharmacological agents (e.g., cytotoxic drugs and corticosteroids),
diabetes, and advanced age. See Hunt et al., in Current Surgical
Diagnosis & Treatment (Way; Appleton & Lange), pp. 86-98 (1988).

[0006] Skin wounds that do not readily heal can cause the subject
considerable physical, emotional, and social distress as well as great
financial expense. See e.g., Richey et al., Annals of Plastic Surgery
23(2):159-65 (1989). Indeed, wounds that fail to heal properly finally
may require more or less aggressive surgical treatment, e.g., autologous
skin grafting. A number of treatment modalities have been developed as
scientists' basic understanding of wounds and wound healing mechanisms
has progressed.

[0007] The most commonly used conventional modality to assist in cutaneous
wound healing involves the use of wound dressings. In the 1960s, a major
breakthrough in wound care occurred when it was discovered that wound
healing with a moist occlusive dressings was, generally speaking, more
effective than the use of dry, non-occlusive dressings. See Winter,
Nature 193:293-94 (1962). Today, numerous types of dressings are
routinely used, including films (e.g., polyurethane films), hydrocolloids
(hydrophilic colloidal particles bound to polyurethane foam), hydrogels
(cross-linked polymers containing about at least 60% water), foams
(hydrophilic or hydrophobic), calcium alginates (nonwoven composites of
fibers from calcium alginate), and cellophane (cellulose with a
plasticizer). See Kannon et al., Dermatol. Surg. 21:583-590 (1995);
Davies, Burns 10:94 (1983). Unfortunately, certain types of wounds (e.g.,
diabetic ulcers, pressure sores) and the wounds of certain subjects
(e.g., recipients of exogenous corticosteroids) do not heal in a timely
manner (or at all) with the use of such dressings.

[0008] Several pharmaceutical modalities have also been utilized in an
attempt to improve wound healing. For example, treatment regimens
involving zinc sulfate have been utilized by some practitioners. However,
the efficacy of these regimens has been primarily attributed to their
reversal of the effects of sub-normal serum zinc levels (e.g., decreased
host resistance and altered intracellular bactericidal activity). See
Riley, Am. Farn. Physician 24:107 (1981). While other vitamin and mineral
deficiencies have also been associated with decreased wound healing
(e.g., deficiencies of vitamins A, C and D; and calcium, magnesium,
copper, and iron), there is no strong evidence that increasing the serum
levels of these substances above their normal levels actually enhances
wound healing. Thus, except in very limited circumstances, the promotion
of wound healing with these agents has met with little success.

[0009] What is needed is a safe, effective, and interactive means for
enhancing the healing of extensive and/or hard-to-heal wounds that can be
used without regard to the type of wound or the nature of the patient
population.

SUMMARY OF THE INVENTION

[0010] The present invention relates to the use of angiogenic or other
growth factors or cytokines expressed by human cells in unencapsulated
preparations (mixed or combined with matrix material or synthetic
biocompatible substances) to be temporarily applied to wounds or defects
in skin or other tissues for the restoration of blood supplying
connective tissue to enable organ-specific cells to reestablish organ
integrity as well as to inhibit excessive scar formation.

[0011] In one aspect, the invention involves a cell preparation useful for
tissue regeneration, e.g., for use in the treatment of skin wounds,
containing one or more cell types that secrete one or more biologically
active substances, admixed with or applied to an extracellular matrix or
matrix material such that the admixture forms a viscous or polymerized
cell preparation. As used herein, the term "admixed" encompasses any
methods of combining, mixing, blending, joining etc. known to those
skilled in the art. The cell types used in the cell preparation of the
invention are allogeneic, optionally mitotically inactivated, and
selected from the group consisting of stromal, epithelial or organ
specific, or blood-derived cells. For example, the cell types may be
differentiated fibroblasts and keratinocytes. In other embodiments, the
cell types may be selected from the group consisting of fibroblasts,
keratinocytes (including outer root sheath cells), melanocytes,
endothelial cells, pericytes, monocytes, lymphocytes (including plasma
cells), thrombocytes, mast cells, adipocytes, muscle cells, hepatocytes,
neurons, nerve or neuroglia cells, osteocytes, osteoblasts, corneal
epithelial cells, chondrocytes, and/or adult or embryonic stem cells.

[0012] The main cell type of connective tissue is the fibroblast. Until
recently, fibroblasts have been dealt with like homogenous
non-differentiating cell populations. However, the fibroblast cell system
in various species, including man, is a stem cell system in which the
fibroblasts terminally differentiate along seven stages, three containing
mitotic and four including post-mitotic cells. See Bayreuther et al.,
Proc. Natl. Acad. Sci. USA 85:5112-16 (1988); Bayreuther et al., J. Cell.
Sci. Suppl. 10:115-30 (1988). In vitro induction of fibroblast
differentiation may be performed by chemical or biological agents, such
as mitomycin C (Brenneisen et al., Exp. Cell. Res. 211:219-30 (1994)) or
growth factors or cytokines (Hakenjos et al., Int. J. Radiat. Biol.
76:503-09 (2000)) such as TGF beta 1, IL-1, IL-6, Interferon alpha. In
vitro induction may also be accomplished by irradiation, e.g., with
γ-rays; X-rays (Bumann et al., Strahlenther. Onkol. 171:35-41
(1995); UV light (Rodemann et al., Exp. Cell. Res. 180:84-93 (1989); or
physical exposure to electromagnetic fields (Thumm et al., Radiat.
Environ. Biophys. 38:195-99 (1999). Moreover, induction of
differentiation may also be accomplished by culture conditions such as
serum starvation, contact inhibition, or the addition of Mitomycin C. See
Palka et al., Folia Histochem. Cytobiol. 34:121-27 (1996).

[0013] To date, the function/biological properties of differentiated
fibroblasts have been poorly studied. The pattern of polypeptide
expression and secretion, however, varies from mitotic to post-mitotic
stages. The respective polypeptides are still being analyzed. See, e.g.,
Francz, Eur. J. Cell. Biol. 60:337-45 (1993).

[0015] In various embodiments, the extracellular matrix or matrix material
used can be collagen, alginate, alginate beads, agarose, fibrin, fibrin
glue, fibrinogen, blood plasma fibrin beads, whole plasma or components
thereof, laminins, fibronectins, proteoglycans, HSP, chitosan, heparin,
and/or other synthetic polymer or polymer scaffolds and solid support
materials, such as wound dressings, that could hold or adhere to cells.
In preferred embodiments, the extracellular matrix or matrix material is
selected from the group consisting of fibrin, fibrin glue, fibrinogen,
fibrin beads, and other synthetic polymer or polymer scaffolds or wound
dressing materials.

[0016] In a further embodiment, the cell types are mitotically
inactivated, e.g., induced to various stages of differentiation. For
example, this mitotic inactivation can be accomplished by the
administration of mitomycin C or other chemically-based mitotic
inhibitors, irradiation with γ-Rays, irradiation with X-Rays,
and/or irradiation with UV light.

[0018] In still further embodiments, the cell types are genetically
engineered to secrete one or more biologically active molecules, such as
at least one angiogenic factor, at least one growth/cytokine factor, or a
combination of at least one angiogenic factor and at least one
growth/cytokine factor. This secretion may be constitutive, or it may be
controlled by gene switching.

[0019] In various other embodiments, the invention also provides methods
of treating tissue defects or wounds by administering the cell
preparations according to the invention to a wound site on a patient in
need of wound treatment. The cell preparation of the invention can be
administered locally (i.e. as a paste) to a wound site on a patient to
temporarily induce tissue regeneration by biological interaction with
surrounding tissues. Alternatively, the cell preparation of the invention
can be administered by spraying the components on a wound site on a
patient to temporarily induce tissue regeneration by biological
interaction with surrounding tissues. The sprayed cell preparation may
result in the formation of a matrix on the wound site.

[0020] In another aspect, the invention involves a method of manufacturing
a cell preparation for tissue regeneration by providing a first
composition containing one or more cells types that secrete one or more
biologically active molecules admixed with thrombin, wherein the cells
types are allogeneic, optionally mitotically inactivated, and selected
from the group consisting of stromal, epithelial/organ specific, and
blood derived cells. The first composition is then combined with a second
composition containing an extracellular matrix or matrix material
containing fibrinogen, wherein the combination of the first and second
compositions forms a viscous cell past suitable for tissue regeneration.
The cell types employed in the cell preparation may naturally secrete the
one or more biologically active molecules, or they may be genetically
engineered to secrete the one or more biologically active molecules.

[0021] In various embodiments, these cell types are differentiated
fibroblasts and keratinocytes, and the biologically active molecule is at
least one angiogenic factor, at least one growth/cytokine factor, or a
combination of at least one angiogenic factor and at least one
growth/cytokine factor. In other embodiments, the cell types may be
mitotically inactivated by administration of mitomycin C or other
chemically-based mitotic inhibitors, irradiation with γ-Rays,
irradiation with X-Rays, or irradiation with UV light.

[0023] In another aspect, this invention provides a kit for the
preparation of a cell preparation for tissue regeneration. This kit
contains a first component containing an extracellular matrix or matrix
material and a second component containing one or more cell types that
secrete a biologically active molecule, such as, at least one angiogenic
factor, at least one growth/cytokine factor, or a combination thereof.
These cell types are allogeneic, mitotically active or inactivated, and
selected from the group consisting of stromal, epithelial/organ specific
and blood derived cells. For example, these cell types may be
differentiated fibroblasts and keratinocytes. The resulting cell
preparation can be in the form of a paste. In various embodiments, the
extracellular matrix or matrix material can be fibrin, fibrin glue,
fibrinogen, fibrin beads, and other synthetic polymer or polymer
scaffolds or wound dressing materials.

[0024] Mitotic inactivation of the cells can be accomplished by
administration of mitomycin C or

[0025] The cell types may naturally secrete the one or more biologically
active molecules or they may be genetically engineered to secrete an
exogenous level of the one or more biologically active molecules.
Secretion may be controlled by gene switching or it may be constitutive.
In one embodiment, the first component contains fibrinogen. In another
embodiment, the first component contains fibrinogen and the second
component contains from about 1×103 cells/μl to about
50×103 cells/μl. The second component also contains
thrombin and can optionally contain a cryoprotectant such as a 10%
glycerol solution, a 15% glycerol solution, and a 15% glycerol and 5%
human serum albumin solution.

[0026] In another aspect, the invention provides methods for using the
kits of the invention to prepare a cell preparation for tissue
regeneration. This method involves administering the first component to a
wound site on a patient in need of treatment and combining the second
component with the first component on the would site, wherein the
combination of the first and second components forms a cell preparation
suitable for tissue regeneration. In one embodiment, the first and second
components are topically administered to the wound site on the patient.
In another embodiment, the first and second components are sprayed onto
the wound site. The components can be sprayed such that they are combined
on the wound or such that they are combined in the air before reaching
the wound.

[0027] In yet another aspect, the invention provides methods of
administering a cell preparation for tissue regeneration to a wound site
on a patient in need of treatment. This method involves the steps of
providing a first component containing an extracellular matrix or matrix
material containing fibrinogen; providing a second component containing
from about 1×103 cells/μl to about 50×103
cells/μl and thrombin, wherein the cells secrete one or more
biologically active molecules, are allogeneic, mitotically active or
inactivated, and selected from the group consisting of stromal,
epithelia/organ specific, and blood-derived cells; combining the first
and second components to form a cell preparation suitable for tissue
regeneration; and administering the cell preparation to the wound site.

[0028] In one embodiment, the first and second components are topically
applied to the wound site. The first component can be applied to the
wound site before or after the second component is applied. In another
embodiment, the first and second components are sprayed on the wound
site. Preferably, the first component is sprayed on the wound site before
the second component is sprayed on the wound site. The first and second
components may be combined on the wound site or they may be combined
before reaching the wound site.

[0029] These cell types may be differentiated or undifferentiated
fibroblasts and keratinocytes. The resulting cell preparation can be in
the form of a paste. In various embodiments, the extracellular matrix or
matrix material can be fibrin, fibrin glue, fibrinogen, fibrin beads, and
other synthetic polymer or polymer scaffolds or wound dressing materials.
Mitotic inactivation of the cells can be accomplished by administration
of mitomycin C or other chemically-based mitotic inhibitors, irradiation
with γ-Rays, irradiation with X-Rays, or irradiation with UV light.
In various embodiments, the cell types are immortalized using at least
one of the following: the 12S and 13S products of the adenovirus EIA
genes, hTERT, SV40 small T antigen, SV40 large T antigen, papilloma
viruses E6 and E7, the Epstein-Barr Virus (EBV), Epstein-Barr nuclear
antigen-2 (EBNA2), human T-cell leukemia virus-1 (HTLV-1), HTLV-1 tax,
Herpesvirus saimiri (HVS), mutant p53, myc, c-jun, c-ras, c-Ha-ras,
h-ras, v-src, c-fgr, myb, c-myc, n-myc, and Mdm2.

[0030] The cell types may naturally secrete the one or more biologically
active molecules or they may be genetically engineered to secrete an
exogenous level of the one or more biologically active molecules.
Secretion may be controlled by gene switching or it may be constitutive.
In one embodiment, the second component can optionally contain a
cryoprotectant including, but not limited to, a 10% glycerol solution, a
15% glycerol solution, and a 15% glycerol and 5% human serum albumin
solution.

[0031] In another aspect, the invention involves a cell preparation for
tissue regeneration containing an extracellular matrix or matrix material
containing fibrinogen admixed with a second component containing from
about 1×103 cells/μl to about 50×103 cells/μl
and thrombin, wherein the cells secrete one or more biologically active
molecules (such as at least one angiogenic factor, at least one
growth/cytokine factor, or combinations thereof) are allogeneic,
mitotically inactivated, and selected from the group consisting of
stromal, epithelia/organ specific, and blood-derived cells.

[0032] These cell types may be differentiated fibroblasts and
keratinocytes. The resulting cell preparation can be in the form of a
paste. In various embodiments, the extracellular matrix or matrix
material can be fibrin, fibrin glue, fibrinogen, fibrin beads, and other
synthetic polymer or polymer scaffolds or wound dressing materials.
Mitotic inactivation of the cells can be accomplished by administration
of mitomycin C or other chemically-based mitotic inhibitors, irradiation
with γ-Rays, irradiation with X-Rays, or irradiation with UV light.
In various embodiments, the cell types are immortalized using at least
one of the following: the 12S and 13S products of the adenovirus E1A
genes, hTERT, SV40 small T antigen, SV40 large T antigen, papilloma
viruses E6 and E7, the Epstein-Barr Virus (EBV), Epstein-Barr nuclear
antigen-2 (EBNA2), human T-cell leukemia virus-1 (HTLV-1), HTLV-1 tax,
Herpesvirus saimiri (HVS), mutant p53, myc, c-jun, c-ras, c-Ha-ras,
h-ras, v-src, c-fgr, myb, c-myc, n-myc, and Mdm2.

[0033] The cell types may naturally secrete the one or more biologically
active molecules or they may be genetically engineered to secrete an
exogenous level of the one or more biologically active molecules.
Secretion may be controlled by gene switching or it may be constitutive.
In one embodiment, the second component can optionally contain a
cryoprotectant including, but not limited to a 10% glycerol solution, a
15% glycerol solution, and a 15% glycerol and 5% human serum albumin
solution.

[0034] The invention also provides methods of using such cell preparations
by providing the first and second components, combining the first and
second components and administering the resulting cell preparation to the
wound site. In various embodiments, the components can be topically
administered to the wound site on the patient or they can be sprayed onto
the wound site. When spray administered, the first and second components
can be combined on the wound site or they can be combined before reaching
the wound site.

[0035] In another aspect, the first and second components of the kits of
the invention is cryopreserved prior to shipping and subsequently thawed
prior to use. Each component may be contained in a separate vial having a
removable screw cap, wherein the vial is sterile and is made of a
material resistant to low temperatures and wherein the removable lid can
be replaced with a spray pump following thawing of the first and second
components prior to use. In one embodiment, the spray pump delivers a
volume of approximately 130 μl per spray. Suitable materials resistant
to low temperatures include, but are not limited to, glass,
polypropylene, polyethylene, and ethylene vinyl acetate (EVA). In some
embodiments, each vial may have a wall thickness of approximately 0.8 ml
and may hold a working volume of approximately 2 ml of the first and
second components. In another embodiment, the vials are sealed within a
pouch or container prior to cryopreservation, wherein the pouch or
container is fabricated of a material capable of withstanding
temperatures ranging from -80° C. to -160° C. and wherein
the pouch or container protects the first and second components from
contamination during cryopreservation, storage, and subsequent thawing.
Preferable, the pouch is waterproof and has a high barrier performance.

[0036] Unless otherwise defined, all technical and scientific terms used
herein have the same meaning as commonly understood by one of ordinary
skill in the art to which this invention belongs. Although methods and
materials similar or equivalent to those described herein can be used in
the practice or testing of the present invention, suitable methods and
materials are described below. All publications, patent applications,
patents, and other references mentioned herein are incorporated by
reference in their entirety. In the case of conflict, the present
specification, including definitions, will control. In addition, the
materials, methods, and examples are illustrative only and not intended
to be limiting.

[0037] Other features and advantages of the invention will be apparent
from the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0038]FIG. 1 is a histogram showing the results of a BrdU cell
proliferation analysis of human primary fibroblasts following 15 and 30
days in culture after gamma irradiation.

[0039]FIG. 2 is a histogram showing adherence of human primary
fibroblasts to a petri dish following gamma irradiation treatments.

[0040] FIG. 3 is a table showing the results of testing for optimal
dilutions of thrombin and fibrinogen for the formulation of a spray
applied fibrin glue matrix.

[0041]FIG. 4 is a histogram comparing the quantity of growth factors
secreted by cell matrices when different doses of cells are sprayed into
individual wells of a 24 well culture dish. The figure also shows growth
factor secretion quantities when the fibrin matrix containing cells is
prepared by simple pipetting (non-sprayed administration) of the
fibrinogen and the thrombin+cell suspensions together.

[0042] FIGS. 5a and 5b are histograms demonstrating that mixing sprayed
keratinocytes and fibroblasts at different ratios, while maintaining a
constant total number of cells, gives rise to variable growth factor
secretion characteristics.

[0043] FIGS. 6a and 6b are pictures of a novel vial used for delivering
and administering the cell preparation of the invention. FIG. 6A is a
cross section of the vial. FIG. 6b is a three-dimensional drawing of the
outside of the vial, which is designed to hold a working volume of 2 ml
of the component.

[0044]FIG. 7 is a histogram comparing growth factor secretion by cells
stored for one week at -160° C. versus cells stored at -80°
C. when using a 10% glycerol solution as a cryoprotectant.

[0047]FIG. 10 is a histogram showing the secretion of the growth factors
GM-CSF and VEGF by cell preparations manufactured at different
productions following either one, four, eight, or twelve weeks of storage
at -80° C. In this experiment, the cell preparation components
were frozen using 15% glycerol+5% human serum albumin as the
cryoprotectant.

[0048]FIG. 11 is a histogram showing growth factor secretion by frozen
cell preparations stored frozen for one week at -80° C. Results
for various keratinocyte:fibroblast ratios and number of cells are shown.

[0050] Generally, an injury to tissue disrupts blood vessels and leads to
extravasation of blood constituents. Blood clotting helps to reestablish
hemostasis and provides a provisional extracellular matrix for cell
migration to the wound. At the wound site, platelets (thrombocytes)
facilitate the formation of a hemostatic plug and also secrete several
mediators of the wound healing process. These mediators include, for
example, molecules such as platelet-derived growth factor that attract
and activate monocytes and fibroblasts.

[0051] Soon after the injury, neutrophils infiltrate the wound and clean
the wound of foreign particles and bacteria. The neutrophils are then
extruded with the eschar or undergo phagocytosis by macrophages.
Monocytes also infiltrate the wound in response to specific
chemoattractants, such as fragments of extracellular matrix proteins,
transforming growth factors β, the monocyte chemoattractant protein
1, and subsequently become activated macrophages. These activated
macrophages release growth factors such as platelet-derived growth factor
and vascular endothelial growth factor, which initiate the formation of
granulation tissue. Macrophages bind through their integrin receptors to
proteins in the extracellular matrix. This binding stimulates macrophage
phagocytosis of any microorganisms as well as of fragments of
extracellular matrix.

[0052] Monocytes, stimulated by adherence to the extracellular matrix,
also undergo metamorphosis into inflammatory macrophages. This adherence
to the extracellular matrix induces monocytes and macrophages to express
colony-stimulating factor 1, tumor necrosis factor β, and platelet
derived growth factor. Other important cytokines expressed by monocytes
and macrophages are transforming growth factor α, interleukin-1,
transforming growth factors β1-3, and insulin-like growth factor 1.
The monocyte- and macrophage-derived growth factors are thought necessary
for the initiation and propagation of new tissue formation in wounds.

[0053] Within hours after the injury, reepithelialization of wounds
begins. Keratinocytes from the wound edges as well as from residual skin
appendages such as hair follicles undergo marked changes in phenotype,
including retraction of intracellular tonofilaments, dissolution of most
intercellular desmosomes, and formation of peripheral cytoplasmic actin
filaments. Furthermore, the hemidesmosomal links between the
keratinocytes and the epidermal basement membrane dissolves, allowing the
movement of keratinocytes.

[0054] Within a few days post injury, the keratinocytes at the margin of
the wound begin to proliferate behind the migrating cells. As this
reepithelialization occurs, the basement-membrane proteins reappear in an
ordered sequence from the margin of the wound outward. Keratinocytes then
revert to their normal phenotype and attach themselves to the
reestablished basement membrane and underlying dermis.

[0055] Within about four days post injury, new stroma begins to infiltrate
the wound. Concomitantly, macrophages, fibroblasts, and blood vessels
also infiltrate the wound.

[0056] Macrophages provide a source of growth factors that stimulate
fibroplasia and angiogenesis. The fibroblasts produce the new
extracellular matrix that supports cell in-growth. The new blood vessels
carry oxygen and nutrients that sustain the cells.

[0057] Growth factors, particularly platelet-derived growth factor and
transforming growth factor β1, are thought to stimulate fibroblasts
of the tissue around the wound to proliferate. In fact, platelet-derived
growth factor has been shown to accelerate the healing of chronic
pressure sores and diabetic ulcers. Basic fibroblast growth factor has
also been used with some success to treat chronic pressure sores.

[0058] However, there are many factors that can lead to abnormal wound
healing. One example occurs with diabetic ulcers. Typically, diabetic
ulcers exhibit multiple biochemical pathologies that can lead to impaired
healing. These ulcers occur in patients who cannot sense and relieve
cutaneous pressure due to some type of diabetic neuropathy. Frequently,
diabetic ulcers become infected because of impaired granulocytic function
and chemotaxis. Patients with diabetic ulcers also experience
inflammation, impaired neovascularization, decreased synthesis of
collagen, increased levels of proteinases, and defective macrophage
function.

[0059] Overall clinical experience using isolated, e.g., recombinant,
growth factors and other mediators to accelerate wound healing has not
met with great success, perhaps because wound repair is the result of a
complex set of interactions between soluble factors, formed blood
elements, extracellular matrix, and cells. Combining various growth
factors at carefully controlled intervals may promote more effective
wound healing.

[0060] The present invention provides stromal, epithelial, and
blood-derived cells, including, but not limited to, fibroblasts,
keratinocytes including follicular outer root sheath cells, endothelial
cells, pericytes, monocytes, lymphocytes including plasma cells,
thrombocytes, mast cells, adipocytes, muscle cells, hepatocytes, nerve
and neuroglia cells, osteocytes, osteoblasts, corneal epithelial cells,
and chondrocytes that are admixed with either a synthetic or natural
extracellular matrix ("ECM") to form a cell preparation that can be used
to improve tissue granulation during wound healing. In one embodiment,
the cells may deliver endogenous angiogenic factors or other growth
factors. In another embodiment, the cells can be genetically engineered
to produce exogenous amounts of the desired factor. Preferably, the cells
are allogeneic. Those skilled in the art will recognize that the cells
employed in the methods and preparations of the invention may include,
but are not limited to, living cells, mitotically inactivated cells,
mitotically activated cells, metabolically inactive cells, lyophilized
cells and/or nonliving cells.

[0061] In particular, fibroblasts and keratinocytes have been shown to
play an important role in cutaneous wound healing. These roles include
stimulating cell migration and proliferation, stimulating extracellular
matrix production, producing growth factors and cytokines, stimulating
angiogenesis, and releasing proteases which dissolve non-viable tissue
and the fibrin barrier.

[0062] Wound healing may be promoted by use of growth and/or angiogenic
factors. For example, one suitable wound healing preparation consists of
two cryopreserved components, i.e., fibrinogen and growth-arrested,
allogeneic human fibroblasts and keratinocytes suspended in thrombin.
After thawing, these components are sprayed sequentially on the wound bed
to form a thin fibrin matrix containing two types of living, but not
proliferating skin-derived cells, which, for several days, interactively
produce growth and angiogenic factors relevant for wound healing (e.g.
VEGF, GM-CSF, bFGF, KGF).

[0063] The cells may be either immortalized or primary cell cultures.
Cells may be immortalized by any method known to those skilled in the
art. A common approach to lengthening the lifespan of a cell is to
transfer a virus or a plasmid that contains one or more immortalizing
genes. Cell immortalization increases the lifespan of a cell, and the
resulting cell line is capable of being passaged many more times than the
original cells. Immortalizing genes are well known in the art. See, e.g.,
Katakura et al., Methods Cell Biol. 57: 69-91 (1998). Immortalizing
proteins or polypeptides include, but are not limited to, the 12S and 13S
products of the adenovirus E1A genes, SV40 small and large T antigens,
papilloma viruses E6 and E7, the Epstein-Barr Virus (EBV), Epstein-Barr
nuclear antigen-2 (EBNA2), human T-cell leukemia virus-1 (HTLV-1), HTLV-1
tax, Herpesvirus Saimiri (HVS), mutant p53, and the proteins from
oncogenes such as myc, c-jun, c-ras, c-Ha-ras, h-ras, v-src, c-fgr, myb,
c-myc, n-myc, and Mdm2. Additionally, cells may become spontaneously
immortalized. A preferred immortalization strategy is by transfer of the
gene encoding telomerase reverse transcriptase (TERT) into the cell such
that TERT was either stably or transiently expressed thereby resulting in
the expression of telomerase activity. Telomerase activity, when
expressed in normal somatic cells, can lead to elongation of the
chromosome tips or protective caps, called telomeres, thereby resulting
in the ability of the telomerase expressing cells to become immortalized
without becoming transformed (See Jiang, et al., Nature Genetics
21:111-14 (1999) and Morales, et al., Nature Genetics 21:115-18 (1999)).

[0066] Telomerase, a unique ribonucleoprotein DNA polymerase, is the only
enzyme known to synthesize telomeric DNA at chromosomal ends using as a
template a sequence contained within the RNA component of the enzyme. See
Greider & Blackburn, 43 Cell 405-413 (1985); Greider & Blackburn, 337
Nature 331-337 (1989); Yu et al., 344 Nature 126-132 (1990); Blackburn,
61 Ann. Rev. Biochem. 113-129 (1992). With regard to human cells and
tissues, telomerase activity has been identified in immortal cell lines
and in ovarian carcinoma but has not been detected in mortal cell strains
or in normal non-germline tissues. See Morin, 59 Cell 521-529, 1989.
Together with TRF analysis, these results suggest telomerase activity is
directly involved in telomere maintenance, linking this enzyme to cell
immortality.

[0067] Expression of the human telomerase catalytic component (hTERT) has
recently been studied in human somatic cells. See Jiang, et al., 21
Nature Genetics 111-114 (1999). Telomerase expression in normal somatic
cells did not appear to induce changes associated with a malignant
phenotype such as abnormal growth control or oncogenic transformation.
The absence of cancer-associated changes was also reported in human
fibroblasts immortalized with telomerase. See Morales, et al., 21 Nature
Genetics 115-118 (1999). It was demonstrated that the introduction of
telomerase into normal human somatic cells does not lead to growth
transformation, does not bypass cell-cycle induced checkpoint controls
and does not lead to genomic instability of these cells. Methods for
detecting telomerase activity, as well as for identifying compounds or
polypeptides that regulate or affect telomerase activity, together with
methods for therapy or diagnosis of cellular senescence and
immortalization by controlling or measuring telomere length and
telomerase activity, have also been described (see PCT International
patent application WO 93/23572). The identification of compounds
affecting telomerase activity provides important benefits to efforts at
treating human disease.

[0068] The cells according to the invention can also be genetically
engineered to produce one or more of biologically active molecules such
that the molecules are constitutively secreted from the cells. By
"constitutively secreted" is meant that the desired biologically active
molecule is continuously expressed by the cells or that the gene is
continually expressed. Alternatively, the cells can be genetically
engineered such that their expression is controlled by gene switching.

[0069] As used herein, the terms "gene switch" and "gene switching" refer
to methods of regulating gene expression. Specifically, expression of a
protein encoded by a gene is controlled by the interaction of certain
regulatory proteins, known as DNA-binding proteins, with a region located
upstream of the gene. Within the promoter region, there are located
several operator regions which contains a specific oligonucleotide
sequence to which these DNA-binding proteins specifically bind. These
proteins can lead either to activation or repression of gene expression.
Thus, they control the regulated expression of genes.

[0070] The regulator protein is encoded by a regulatory gene located
elsewhere on the chromosome. The interaction of regulator and operator is
affected by the presence or absence of particular chemical factors
(inducers). Thus, in normal circumstances the regulator is expressed,
thereby binding the operator and inhibiting expression of the gene, until
a need for the particular protein encoded by the gene is indicated by the
appearance in the environment of a specific inducer which interacts with
the regulator to inhibit binding to the operator, thus allowing
expression of the gene.

[0071] For example, an enzyme, which acts upon a sugar molecule is not
required unless that sugar is present and, therefore, in the absence of
the sugar, the regulatory gene expresses the regulator protein, which
binds the gene operator and inhibits expression of the enzyme. The sugar
itself acts as the inducer, which then interacts with the regulator to
prevent its binding to the operator thus allowing expression of the
enzyme. Digestion of the sugar by the enzyme removes it from the
environment allowing the regulator to return to its normal mode and act
normally to inactivate enzyme expression.

[0072] Such a regulatory mechanism can be viewed as a switching
arrangement which switches gene expression on and off as dictated by the
chemical content of the environment. Gene switching systems of the type
described are best known in bacteria and many of the proteins and their
target DNA binding sites are known in considerable detail. The regulator
proteins usually bind as dimers to operators, which exhibit a two-fold
symmetry. The specificity of the regulator/promoter interaction is
determined by the sequence specific interaction of specific amino acids
of the regulator with the operator DNA. In some systems interactions have
been subject to detailed biochemical analysis as well as high resolution
X-ray crystallography. The best-characterized class of DNA binding
proteins exhibit a common helix-turn-helix motif with some degree of
amino acid sequence homology. It is clear that the critical DNA binding
domain of the regulator is contained within the helix-turn-helix region.

[0073] In eukaryotes it has been shown that control of gene expression is
also regulated by the interaction of specific protein factors binding to
DNA sequences close to the promoter region of genes. A number of factors
have been isolated from yeast and mammalian cells and have been shown to
interact with specific sequence motifs in a sequence-specific manner
similar to that observed in bacterial systems. Characterization of some
of these factors has revealed a new "finger" motif, which may be involved
in the sequence specific binding of proteins.

[0074] Moreover, it has been demonstrated that eukaryotic gene expression
can be controlled through the use of bacterial repressor molecules in
eukaryotic cells. In these experiments bacterial operator sequences have
been inserted close to the promoters of mammalian genes. Cell lines have
been created which express the bacterial repressor. Control of expression
of the target eukaryotic genes with operator insertions by repressor
molecules has been demonstrated using transient expression assays. In
these experiments not only repression of gene expression by the lac
repressor has been demonstrated but also induction of gene expression,
that is, relief of repression, using IPTG (isopropyl thiogalactoside).

[0075] Therefore, detailed knowledge and manipulation of bacterial protein
DNA/interactions can be used to control expression in mammalian cell
cultures. Gene switching techniques are described, for example in U.S.
Pat. No. 6,010,887, which is incorporated herein by reference. Those of
ordinary skill in this art will recognize that other methods of gene
switch regulation may also be employed in the methods and compositions of
the invention.

[0076] Although non-genetically modified cells may be used in accordance
with the invention, in one preferred embodiment, the isolated cells are
genetically engineered. The cells can be genetically engineered to
secrete one or more biologically active molecules including, but not
limited to, one or more cytokines, growth factors, and/or angiogenic
factors, or a combination thereof. Examples of such biologically active
molecules are provided in Table 1.

[0080] Control of the delivery of the secreted biologically active
molecule can be achieved by any method known to those skilled in the art.
For example, the expression of multiple gene products may be controlled
by a single promoter system. Alternatively, the expression of multiple
gene products may be controlled by multiple promoter systems, with each
promoter system regulated either constitutively, by gene switching or by
some combination of both. Further, control over the secretion of a
particular biologically active molecule may be accomplished by
up-regulating wild-type gene expression.

[0082] Moreover, one skilled in the art will recognize that any other
method suitable for delivering an exogenous biologically active molecule
into the cell types may also be employed in accordance with the
invention.

[0083] Using any of the above-mentioned transfection methods, control over
the secretion of a variety of biologically active molecules may be
achieved by employing any number of cell types secreting the various
biologically active molecules.

[0084] Additionally, the cell types of the present invention may also be
either mitotically active or mitotically inactive. The term "mitotically
active" is used to describe cells that actively undergo mitosis.
Conversely, "mitotically inactive" is used to describe cells that do not
actively undergo mitosis. Mitotically inactive cells may be growth
arrested by any means known in the art. By way of non-limiting example,
the cells may be growth arrested by chemical means, such as, for example,
by the administration of mitomycin C. Additionally, the cell types may be
growth arrested by exposure to UV light, X-Ray, or gamma (γ)
radiation. In one preferred embodiment, the cell types are growth
arrested by exposure to gamma radiation. It is important to note that,
e.g., mitotically inactivated, human fibroblast cells terminally
differentiate and thereby change the pattern of polypeptide expression
and secretion (Francz, Eur. J. Cell. Biol. 60:337-45 (1993)). As a
further example, keratinocyte differentiation usually depends on culture
conditions (including, for example, the composition of culture media, the
Ca2+-concentration, and whether the cells are cultured at the
air-liquid interface), however, keratinocytes may also be induced to
differentiate, e.g., by mitomycin C.

[0085] The cell types according to the invention may be autologous,
allogeneic, or xenogeneic. Most preferably, the cell types used in the
present invention are allogeneic. Xenogeneic cells can be isolated for
example from transgenic animals expressing molecules of interest.

[0086] Stromal cells including, for example, fibroblasts, can be isolated
by any method known to those skilled in the art. For example, fibroblasts
may be derived from organs, such as skin, liver, and pancreas. These
organs can be obtained by biopsy (where appropriate) or upon autopsy.
Specifically, sufficient quantities of fibroblasts can be obtained rather
conveniently from breast reduction, foreskin, or any appropriate cadaver
organ.

[0087] Fibroblasts can be readily isolated by disaggregating an
appropriate source organ or tissue. By "source organ or tissue" is meant
the organ or tissue from which the cells are obtained. Disaggregation may
be readily accomplished using techniques known to those skilled in the
art. Examples of such techniques include, but are not limited to
mechanical disaggregation and/or treatment with digestive enzymes and/or
chelating agents that weaken the connections between neighboring cells
thereby making it possible to disperse the tissue into a suspension of
individual cells without appreciable cell breakage. Specifically,
enzymatic dissociation can be accomplished by mincing the tissue and
treating the minced tissue with any of a number of digestive enzymes,
either alone or in combination. Suitable enzymes include, but are not
limited to, trypsin, chymotrypsin, collagenase, elastase, hyaluronidase,
DNase, pronase, and/or dispase. Mechanical disruption can be accomplished
by a number of methods including, but not limited to, the use of
grinders, blenders, sieves, homogenizers, pressure cells, insonators or
trituration. See Freshney, Culture of Animal Cells. A Manual of Basic
Technique, 2d Ed., A. R. Liss, Inc., New York, 1987, Ch. 9, pp. 107-26.

[0088] Once the source tissue has been reduced to a suspension of
individual cells, the suspension should be fractionated into
subpopulations from which the fibroblasts and/or other stromal cells
and/or elements can be recovered. Fractionation may be accomplished using
standard techniques for cells separation including, but not limited to,
cloning and selection of specific cells types, selective destruction of
unwanted cells (negative selection), separation based upon differential
cell agglutinability in the mixed population, freeze-thaw procedures,
differential adherence properties of the cells in the mixed population,
filtration, conventional and zonal centrifugation, centrifugal
elutriation (counter-streaming centrifugation), unit gravity separation,
countercurrent distribution, electrophoresis and fluorescence-activated
cell sorting. See Freshney, Culture of Animal Cells. A Manual of Basic
Techniques, 2d Ed., A. R. Liss, Inc., New York, 1987, Ch. 11 and 12, pp.
137-68. Those skilled in the art will recognize that other suitable cell
fractionation technique(s) can also be used.

[0089] Preferably, the isolation of fibroblasts is accomplished by
explantation of skin pieces according to the method of Sly and Grubb. See
Sly et al., Methods Enzymol. 58:444-50 (1979).

[0090] Fibroblasts obtained from different source organs or tissues
(including, e.g., skin, liver, and pancreas) can be employed in the
methods and compositions of the invention. Moreover, those skilled in the
art will recognize that any such fibroblasts can be genetically
engineered to secrete differing amounts of a biologically active molecule
or molecules.

[0091] As used herein, the term "cell preparation" refers to the mixture
resulting from the combination of a preparation of cells that secrete one
or more biologically active molecules and a preparation of the
extracellular or other matrix materials. For example, a cell preparation
according to the invention results from the combination of a
thrombin/cell preparation and a fibrinogen preparation. In some
embodiments, the resulting mixture results in polymerization, thereby
producing a cured matrix. Alternatively, the resulting combination may
produce a highly viscous, non-cured matrix. The resulting cell
preparation can be in the form of a paste. Those skilled in the art will
recognize that as used herein, the term "cell preparation" encompasses a
spectrum of mixtures ranging from a viscous, non-cured mixture (i.e. a
paste) to a polymerized, cured matrix. Differences in the concentration
of each preparation as well as the culture conditions can influence the
viscosity and/or the degree of polymerization of the resulting cell
preparation. For example, in one embodiment of the invention, the
combination of the fibrinogen and the thrombin/cell preparations are
spray administered to a wound site to form an irreversible, polymerized
cell matrix. However, those skilled in the art will also recognize that
other combinations may result in a viscous, non-cured cell matrix.

[0092] To create the cell preparation according to the invention, the cell
types or genetically engineered cell lines are preferably admixed or
combined with a supporting biological or synthetic extracellular matrix
or matrix material (ECM). One skilled in the art will recognize that the
term "ECM" refers to the noncellular material distributed throughout the
body of multicellular organisms. The ECM is comprised of diverse
constituents such as glycoproteins, proteoglycans, complex carbohydrates,
and other molecules. Major functions of the ECM include, but are not
limited to, providing structural support, tensile strength or cushioning;
providing substrates and pathways for cell adhesion and cell migration;
and regulating cellular differentiation and metabolic function. ECM
proteins include, for example, collagens, elastin, fibronectin, laminin,
proteoglycans, vitronectin, thrombospondin, tenascin (cytoactin),
entactin (nidogen), osteonectin (SPARC), anchorin CII, chondronectin,
link protein, osteocalcin, bone sialoprotein, osteopontin, epinectin,
hyaluronectin, amyloid P component, fibrillin, merosin, s-laminin,
undulin, epilligrin, and kalinin. Preferred ECM proteins for use
according to this invention include collagen, alginate, agarose, fibrin,
fibrin glue, fibrinogen, laminins, fibronectins, HSP, chitosan, heparin
and/or other synthetic polymer or polymer scaffolds.

[0093] Cell density and the concentration of the extracellular matrix may
be varied for the desired clinical application. For example, certain
wounds may require greater or lesser cell densities and/or different
consistency preparations. The resulting cell preparations can be in the
form of a cell paste or in the form of a cured matrix. Determination of
the appropriate cell density and concentration of the ECM is within the
routine skill of those in the art. The cell suspension can come from one
cell type or can be comprised of a mixture of different cell types. For
example, the cell mixture may include 50% of keratinocytes and 50% of
fibroblasts. However, the ratio of keratinocytes to fibroblasts may be
varied, e.g. 1:1, 1:4, 1:9, 1:24 etc. without impairing the formation or
the function of the cell preparation. Alternatively, the cell mixture may
contain only keratinocytes or only fibroblasts (i.e., the ratio of
keratinocytes to fibroblast may be 1:0 or 0:1). Moreover, the suspension
may be comprised of more than two different cell types. The percentages
of each cell type in the cell suspension may vary depending on the
intended use for the cell preparation. The cell types can also be
pre-induced or co-cultured in vitro in order to optimize the healing
response on the wound. For example, fibroblasts can be pre-incubated with
TGF-beta (from 0.1 to 30 ng/ml of medium) for 1 to 21 days prior to wound
application.

[0094] The cell preparation of the invention is made from two components.
The first, referred to herein as "component #1" is the fibrinogen
component. The second, referred to herein as "component #2" is the
cells+thrombin component. Component #2 can optionally contain a
cryoprotectant. The cell preparation of the invention is formed by the
coagulation of plasma proteins (including fibrinogen) in the presence of
thrombin. This coagulation is chiefly due to the formation of a
polymerized fibrin network, which imitates the formation of a blood clot.
Thrombin converts fibrinogen to fibrin by enzymatic cleavage. Calcium
accelerates the proteolytic activity of thrombin. In fact, the
combination of fibrinogen and thrombin results in a "polymerization
reaction". Upon mixing of these materials, the cells become entrapped in
the resulting cell preparation, which may be a paste or a cured matrix,
depending on the concentrations of all components, the number of cells,
the culture conditions, etc. Additionally, in any of the cell
preparations of the invention, any or all of the components may also
contain additional proteins or chemicals, which do not affect the
formation or function of the cell preparation, such as proteins (L e.
Albumin), proteinase inhibitors (i.e., Aprotinin), polyethylene glycol
(PEG), polyvinyl alcohol (PVA), and other molecules typically used as
stabilizers for cell preparations. In other embodiments of the invention,
the first component of the cell preparation may contain thrombin and the
second component may contain cells plus fibrinogen. Those skilled in the
art will recognize that, in this embodiment, the combination of component
#1 and component #2 will also result in a cell preparation useful for
tissue regeneration. Moreover, in other embodiments, the cell
preparations suitable for tissue regeneration may result from "synthetic
polymerization" rather than from polymerization following the interaction
of fibrinogen and thrombin. In this embodiment, the cells are mixed with
a polymerization agent, either before or after application to the wound
site. Once polymerization occurs, a cell preparation suitable for tissue
regeneration may be formed.

[0095] While the compositions, cell preparations, kits, and/or methods
described herein refer to the use of a first component containing
fibrinogen and a second component containing cells+thrombin, other
components may also be used. Examples of such components include the
thrombin component #1 and the cells+fibrinogen component #2 as well as
the components leading to synthetic polymerization, which are discussed
in detail above. Those skilled in the art will recognize that any of the
compositions, cell preparations, kits, and/or methods described herein
using the fibrinogen component #1 and the cells+thrombin component #2 may
also employ any other combination of components which result in a cell
preparation for tissue regeneration in which the cells become entrapped
in the resulting paste or matrix, without deviating from the nature of
the invention.

Methods of Administration

[0096] In one preferred embodiment, the invention involves a combination
of human allogeneic fibroblast and keratinocyte cell lines admixed with
ECM materials to form a viscous cell paste to adhere to a wound. In this
embodiment, the cell lines are preferably not genetically engineered. The
cell lines may be mitotically inactivated by any means known to those
skilled in the art. Preferably, the resulting paste is both biodegradable
and biocompatible. The paste may be applied to the wound as needed, for
example, once weekly. Application of the cell paste according to this
embodiment facilitates the induction of granulation tissue and the
stimulation of wound closure.

[0097] As previously described, immortalized fibroblast and keratinocyte
cell lines would also be preferred embodiments. The preferred
immortalization method would be through directly adding the gene for TERT
into the primary human keratinocyte and fibroblast cells such that the
TERT gene is constitutively expressed. In addition, a transient
immortalization using a protein domain transport sequence (TAT, VP22,
MTS, etc. attached to the TERT protein might be more preferable in that
the gene is not permanently inserted into the immortalized cell but is
instead added as a fusion protein to the growth medium. In this way, the
cell line could be continuously expanded, banked, and screened for stable
properties (growth rate, factor secretion, etc.), without the continual
need for the revalidation of new primary cell sources. Cell lines
immortalized in this way would preferentially be mitotically inactivated
before application to the wound or tissue repair site as a paste, cell
preparation, biological matrix mixture, or as attached or adsorbed to a
wound dressing.

[0098] The present invention has human clinical and veterinary
applications. The cell preparation of the invention can be used to treat
humans and non-human animals, including, a non-human primate, mouse, rat,
dog, cat, pig, sheep, cow, or horse. The cell preparation according to
the invention can be used for tissue regeneration such as, e.g., in skin
wound treatment or in treatment of peritonitis.

[0099] For example, the cell preparation of the invention can be
incorporated into other pharmaceutical compositions suitable for
administration. Such compositions can comprise the cell preparation and
an additional acceptable carrier. As used herein, "biologically
acceptable carrier" is intended to include any and all solvents,
dispersion media, coatings, antibacterial, and antifungal agents,
isotonic and absorption delaying agents, and the like, compatible with
biologics administration. Suitable carriers are described in the most
recent edition of Remington's Pharmaceutical Sciences, a standard
reference text in the field, which is incorporated herein by reference.
Preferred examples of such carriers or diluents include, but are not
limited to, water; saline; dextrose solution; human serum albumin; HBSS
and other buffered solutions (including those with and without Ca++
and Mg++) known to those skilled in the relevant arts; and basal
media. Liposomes and non-aqueous vehicles such as fixed oils may also be
used. The use of such media and agents for pharmaceutically active
substances is well known in the art. Except insofar as any conventional
media or agent is incompatible with the active compound, use thereof in
the compositions is contemplated. Supplementary active compounds can also
be incorporated into the compositions.

[0100] The pharmaceutical compositions can be included in a container,
pack, kit, or dispenser together with instructions for administration.

[0101] The dosage regimen is selected in accordance with a variety of
factors including species, age, weight, sex, and medical condition of the
patient; type and severity of the condition to be treated; the route of
administration; and the particular cells employed. An ordinarily skilled
physician or veterinarian can readily determine and prescribe the
effective amount required to prevent, counter, or arrest the progress of
the condition.

[0102] The cell preparation of the invention can be administered topically
to the wound in need of treatment. The thrombin+cell component (component
#2) can be admixed with the fibrinogen component (component #1) before,
during, or after application to the wound. In one embodiment, the
components are applied to the wound site by simple pipetting or by
co-extruding components from a tube or syringe applicator. Each
components may also be applied in conjunction with a sterile gauze
dressing. Moreover, those skilled in the art will recognize that the
order of topical administration of the components can be varied (e.g.
thrombin+cells component followed by fibrinogen component or fibrinogen
component followed by thrombin+cells component).

[0103] The cell preparation of the invention can also be administered by
spraying the components of the preparation onto the wound area. Spray
pumps suitable for use in administering the cell preparation of the
invention include those used for other medical applications, including
nasal and throat sprays. Additionally, the cell preparation could be
applied using a spray generated by compressed inert gasses rather than
using a spray pump. For example, small canisters of medical grade inert
gasses such as air and/or nitrogen can be used. The pressure of the gas
can be used to propel fluids through a small orifice, thereby generating
a fine mist spray. The pressure can act directly on the fluid or a
pressure drop can pull fluid out into an air stream, which eventually
would become a spray.

[0104] In another embodiment, the cell preparation of the invention may be
spray administered as a two component product that is applied to a
chronic ulcer in a sequential, two-step process. In this embodiment, the
first component is a suspension of fibrinogen in HBSS (without Ca++
and Mg++) and the second component is a mixture of cells (including
fibroblasts and keratinocytes), thrombin and cryoprotectants in HBSS
(with Ca++ and Mg++). In this embodiment, the two components of
the cell preparation are mixed together on the target wound area. Both
components are applied to the chronic ulcer or wound using a spray
applicator, such as a spray pump. For example, a spray pump may be used
to deliver precise doses of both components during treatment of an ulcer
or wound. The actual design of the spray pump used may vary depending on
the manufacturer. Examples of suitable spray pumps include for example
nasal and throat spray. The spray pump is manufactured of a material that
can be sterilized using conventional techniques to avoid contamination of
either component of the cell preparation.

[0105] Sprayed doses can range from about 50 μl to about 150 μl per
spray, preferably from about 100 μl to about 150 μl per spray, most
preferably about 130 μl per spray. To allow for concentrated
application and for more precise delivery of the product, the spray pump
should have a spray actuator mechanism that produces a narrow spray
diameter rather than a large diameter spray. The spray actuator mechanism
is the "arm" that orients and generates the spray via the orifice size.
The area of the chronic ulcer or wound surface area covered by each spray
depends directly on the distance of the actuator from the target. For
example, the closer the spray actuator mechanism is to the target area,
the smaller the surface area covered per spray. Likewise, the further
away from the target area the spray actuator mechanism is, the larger the
surface area covered per spray.

[0106] Preferably, the surface area covered ranges from about 11 cm2
to about 14 cm2. In one preferred embodiment, the distance between
the spray nozzle and the target is approximately 6 cm. At this distance,
using a narrow diameter spray actuator mechanism, one spray will cover a
wound surface area of approximately 12.6 cm2.

[0107] The number of cells landing on the target area of the patient
(i.e., the number of cells per cm2 of patient) will vary depending
on the concentration of each of the components and the ratios of
keratinocytes to fibroblasts used in component #2. Those skilled in the
art will recognize that the concentration of cell in the second component
of the cell preparation can be varied from about 1×103
cells/μl to about 50×103 cells/μl. For example, in some
embodiments, the number of cells/μl of the cell preparation component
#2 can range from about 5×103 cells/μl to about
20×103 cells/μl. Thus, if two sprays of approximately 130
μl/spray are administered to a patient, approximately about
1.3×106 to about 5.2×106 cells are administered to
the patient.

[0108] Typically, the first component (fibrinogen) is sprayed onto the
target followed by spraying the second component (thrombin+cells). The
order of spraying of the components can be reversed. However, it is
preferable to first apply the fibrinogen component and then subsequently
apply the thrombin+cells component, which may optionally contain a
cryoprotectant. Once the cells+thrombin component is sprayed onto the
fibrinogen component, the two components will begin to gel, cure or
polymerize almost immediately, allowing an equal distribution of cells on
the target area. When the cells+thrombin component is applied first
followed by the fibrinogen component, the resulting lag time allows the
cells to migrate on the wound site due to the effects of gravity, which
might cause the cells+thrombin component to "run" or "drip" after
application, depending on the volume applied. This, in turn, could
potentially lead to an unequal distribution of cells upon application of
the fibrinogen component. Since the subsequent application of the
fibrinogen component leads to polymerization, this could result in the
formation of an uneven cell preparation.

[0109] In another embodiment, the cell preparation components can be
administered as a spray that is mixed in the air prior to reaching the
target area. In such an embodiment, two separate components could be
sprayed at the same time using one or two spray actuator mechanisms. The
spray mists of each component would then combine in the air, thereby
initiating polymerization before the cell preparation reaches the target
area (i.e., the wound site). This embodiment would make the cell
preparation of the invention easier to apply, as it requires a single
spray to apply the cells and to initiate fibrin polymerization.

Cryopreservation

[0110] In some embodiments, component #2 cells may be cryopreserved prior
to use in the cell preparation of the invention. In such embodiments,
component #2 contains cells+thrombin+a cryoprotectant. Those skilled in
the art will recognize that, the terms "cryoprotectant" and
"cryopreservant" are used interchangeably herein and cover agents used to
achieve cryopreservation. Suitable cryoprotectants include, e.g.,
glycerol, ethylene glycol, and dimethyl sulfoxide ("DMSO"). In various
embodiments, the cryoprotectant includes, but is not limited to a 10%
glycerol solution, a 15% glycerol solution, and a 15% glycerol and 5%
human serum albumin (HSA) solution. Those skilled in the art will
recognize that any other cryoprotectants and cryoprotectant
concentrations known in the art may also be used. The cryoprotectants
used in the instant invention can be included in the buffers containing
component #2 of the cell preparation. In this protocol, no extra proteins
need to be added. Likewise, the cells are not frozen in biological media.
By choosing a cryoprotectant with low or no toxicity, there is no need to
wash away or otherwise remove the cryoprotectant from the cells prior to
use. This allows direct application of the cell preparation after
thawing.

[0111] Cryopreservation facilitates shipping and long-term storage of the
components of the cell preparation of the invention. Cryopreserved cells
(in component #2) are stored at a temperature ranging from about
-70° C. to about -196° C. (if liquid nitrogen is used). For
example, the cryopreserved cells may be stored in a -80° C.
freezer or in the vapor phase of liquid nitrogen at -160° C.

[0112] In one preferred cryopreservation protocol, a vial containing the
cell+thrombin+cryoprotectant mixture (component #2) is closed with a
screw-on closure in a sterile manner. The closed vial is then packaged
(hermetically sealed) inside a pouch fabricated of a material that can
resist temperatures ranging from -70° C. to -196° C., which
is the temperature found in the liquid nitrogen vapor phase. Other
storage temperatures between -120° C. and -160° C. can be
found in the liquid nitrogen vapor phase. Vials or pouches containing
vials are then placed inside a controlled rate freezer employed for the
freezing of biological cells and tissues in a special rack designed to be
inserted inside the controlled rate freezer. Typically, the vials or
pouches are stored upright and aligned in several rows, such that there
is space between each row. The rows are aligned parallel to the flow of
the liquid nitrogen vapor that passes through the chamber to cool the
samples. The use of such pouches helps to prevent contamination of the
components during freezing and thawing of component #2.

[0113] After loading the samples into the chamber, a freeze cycle is
initiated, as follows:

[0114] 1. Beginning at room temperature or
20° C., the chamber is cooled at -2° C. per minute until
4° C.

[0115] 2. The chamber is then held at 4° C. for 52
minutes to stabilize all samples at 4° C.

[0116] 3. The chamber is
then cooled at -1° C. per minute until -5° C.

[0117] 4. The
chamber is then cooled at -3° C. per minute until -12° C.

[0118] 5. The chamber is then cooled at -7.5° C. per minute until
-20° C.

[0119] 6. The chamber is then held at -20° C. for
15 minutes.

[0120] 7. The chamber is then cooled at -1° C. per
minute until -80° C.

[0121] 8. The chamber is then held at
-80° C. for 60 minutes.

[0122] The pouches are then removed from the freezer and stored at either
-160° C. or -80° C. until the time of use.

[0123] Those skilled in the art will recognize that the exact method used
in the cooling cycle may be changed or improved upon as necessary. In
general, it is well known that cells or tissues should be cooled at a
rate ranging from about -0.2° C./minute to about -5°
C./minute. Moreover, cooling rate ranges of about -0.5° C./minute
to about -2° C./minute are optimal for most cases.

[0124] The cooling rate is especially critical as the temperature is
lowered to the freezing point of the cryoprotectant solution used. In the
preferred embodiment described herein, cooling is accelerated around the
freezing point of the solution. This global, rapid drop in chamber
temperature is intended to induce the phase change by generating a
thermal instability that would induce ice seeding. It is desirable to
control the seeding of the phase transition process in order to initiate
the phase transition of all samples at the same time. Seeding of the
phase transition process may be controlled by several methods, including:
creation of a point thermal instability by either (1) touching a sample
with a metal probe chilled in liquid nitrogen or (2) ejecting a cooled
liquid (e.g. liquid nitrogen) at the sample. (See U.S. Pat. Nos.
6,347,525; 6,167,710; and 5,981,617, each of which are incorporated
herein by reference). Those skilled in the art will recognize that other
means of controlling the phase transition can also be used. For example,
any available technique that creates an instability in the supercooled
solution such as mechanical stimulation (e.g. a pulsed vibration) can be
used.

[0125] In some embodiments, the cell preparation of the invention can be
delivered as a kit contained in a single package made from an aluminum
foil laminate pouch that resists low temperatures. Inside this outer
pouch are two smaller pouches, the first with a vial containing component
#1 (fibrinogen) and the second with a vial containing component #2
(cells+thrombin+cryoprotectant). Alternatively, vials for components #1
and #2 can be packaged together in a single pouch or case. These inner
pouches are also made from a temperature resistant material. Examples of
suitable materials for these pouches include, but are not limited to, an
aluminum foil laminate pouch, coated paper, LDPE, Surlyn®, and a
Kapton® laminate pouch (Steripack, Ireland). Those skilled in the art
will recognize that any other suitable material may also be used to make
these pouches. The material used to manufacture the pouch should have a
low seal initiation temperature, high barrier performance, and good
chemical resistance. Moreover, it should be suitable for irradiation
sterilization to avoid contamination of the cell preparation components.
An example of the composition and typical properties of a suitable pouch
are provided in Tables 3 and 4.

[0126] The sealing parameters for a suitable pouch will depend on the
particular sealing equipment used and its compatibility with the sealant
layer of the secondary substrate. However, typical parameters for sealing
can include: temperature=100-140° C.; dwell time=0.30-0.75 sec;
and pressure=60-80 psi.

[0127] In another embodiment, the vials may be sealed in a rigid,
transparent container, fabricated by using a polymer resistant to
temperatures below -70° C. to as low as -196° C. A variety
of different types of vials may be used for freezing components #1 and
#2. In one preferred embodiment, a novel vial (5) such as that shown in
FIG. 6A and FIG. 6b is employed. This vial is used to freeze down the
components of the cell preparation of the invention. Upon thawing, the
screw-on cap (4) closing the vial can be replaced with a spray pump
applicator. In one embodiment, bottle may be made of polypropylene, which
is resistant to the low temperatures employed in the cryopreservation
protocol. The wall thickness of this vial (1) should be approximately 0.8
mm to facilitate heat/cold transfer across the wall, which is important
for both the freezing and thawing processes. Additionally, the vial can
be designed to stand upright after a spray pump has been screwed on, and
the bottom of the vial (2) is conical (3) to facilitate emptying of the
contents.

[0128] One spray pump designed to be used with this type of vial is
manufactured by the company Valois. Each spray delivers a 130 μl
volume of product. The spray actuator, which is the "arm" that orients
and generates the spray via the orifice size, can be modified so that the
"arm" can be oriented in directions other than the horizontal position,
to aid in topical administration by allowing spray application onto a
horizontal surface without tipping the bottle. In another embodiment,
other spray pump designs can be employed which allow the spray to
function when the bottle is inverted.

[0129] The cryopreserved components of the cell preparation of the
invention can be shipped, stored, and/or used as follows. The components
may be shipped as a kit frozen on dry ice at a temperature of about
-70° C. to about -80° C. The pump may be shipped at room
temperature. Upon arrival, the kit should be stored in a -80° C.
freezer or at -160° C. until use. To use, the outer pouch of the
kit is opened and the two smaller pouches containing component #1
(fibrinogen) and #2 (cells+thrombin+cryoprotectant) are removed and
thawed in a water bath that is warmed to a maximum of 37° C. Those
skilled in the art will recognize that the pouches serve to prevent water
in the water bath from contaminating the components of the cell
preparation. Once the contents of the vials are thawed, the pouches can
be removed from the water bath, disinfected (if desired), and opened. The
screw-on top from each vial is then removed and replaced with a screw-on
spray applicator, and the cell preparation components are then ready for
patient application.

[0130] The invention will be further described in the following examples,
which do not limit the scope of the invention described in the claims.

Example 1

Isolation of Keratinocytes and Fibroblasts

[0131] Keratinocytes and fibroblasts may be isolated after splitting the
epidermis from the dermis using an enzyme such as dispase or thermolysin.
The isolated epidermis can be incubated with trypsin to obtain a single
cell suspension of keratinocytes, which can then be plated onto culture
dishes and amplified to create a bank of primary keratinocytes. The
isolated dermis can be incubated with a dissociation enzyme such as
collagenase to obtain fibroblast single cell suspensions ready to be
plated and amplified or minced and dispatched onto a culture plate, and
cultured until fibroblasts have migrated from the tissue. These cells can
then be collected after trypsin treatment and further amplified to
establish a fibroblast cell bank. Primary human keratinocytes and
fibroblasts isolated in this manner can be used for the preparation of
cell and fibrin admixtures.

[0132] The isolation of fibroblasts may also be carried out as follows:
fresh tissue samples are thoroughly washed and minced in Hank's balanced
salt solution (HBSS) in order to remove serum. The minced tissue is
incubated from 1 to 12 hours in a freshly prepared solution of a
dissociating enzyme such as trypsin. After such incubation, the
dissociated cells are suspended, pelleted by centrifugation and plated
onto culture dishes. All fibroblasts will attach before other cells,
therefore, appropriate stromal cells can be selectively isolated and
grown. The isolated fibroblasts can then be grown to confluency, and
serially cultured or stored frozen in liquid nitrogen (see, Naughton et
al., 1987, J. Med. 18(3&4):219-250). Fibroblasts or subpopulations of
fibroblasts such as dermal papilla cells or myofibroblasts can be
isolated from explant outgrowth culture. Once isolated, the stromal cells
are ready for admixture with an extracellular matrix (e.g. fibrin) paste.

Example 2

Composition of the Components of the Cell Preparation of the Invention

[0133] Component #1

[0134] This component can be made by performing a four-fold dilution of
the Sealer Protein Solution in the Tisseel VH Fibrin Sealant (Baxter).
After dilution, the concentration of the components in the Sealer Protein
Solution was as follows:

[0135] Fibrinogen: 18.75 mg/ml-28.75 mg/ml

[0136] Fibrinolysis Inhibitor (Aprotinin): 750 KIU/ml

[0137] Polysorbate: 0.05 mg/ml-0.1 mg/ml

[0138] Sodium Chloride: 0.5 mg/ml-1.0 mg/ml

[0139] Trisodium Citrate: 1.0 mg/ml-2.0 mg/ml

[0140] Glycine: 3.75 mg/ml-8.75 mg/ml

[0141] The Sealer Protein Solution was diluted in Hank's Buffered Saline
Solution (HBSS) without Ca2+ or Mg2+. The presence of these two
ions induced the formation of precipitates during the freezing and
thawing process.

[0142] Those skilled in the art will recognize that the Tisseel Sealant
can be supplemented with other commercially available fibrinogen and
aprotinin preparations to achieve similar results. Moreover, fibrinogen
may also be diluted to other concentrations, depending on the mode of
administration, to enhance the polymerization characteristics.

[0143] Component #2

[0144] This cryoprotected component can be made by mixing the following:

[0145] Following dilution, the thrombin solution obtained from Baxter
contributes the following to the cryoprotected solution:

[0146]
Thrombin: 50 IU/ml

[0147] Total protein: 4.5-5.5 mg/ml

[0148] Sodium
Chloride: 0.8-1.2 mg/ml

[0149] Glycine: 0.24-0.36 mg/ml

[0150]
CaCl2: 4 μmol

[0151] HBSS with Ca2+ and Mg2+ was the chosen diluent.

[0152] The desired cell mixture (ratio) and concentration was resuspended
in the cryoprotected solution to obtain the component #2. Various
keratinocyte:fibroblast ratios have been considered, including 1:1, 1:3,
1:4, 1:9, to as high as 1:50. Moreover, various final cell concentrations
in component #2 considered have been 1 million, 2.5 million, 5 million,
10 million, 20 million, and 50 million cells/ml (final concentration).

[0154] The rat fibroblast cell line (CRL 1213), the FGF1-transfected rat
fibroblast cell line (1175/CRL 1213), the human telomerase immortalized
fibroblast line (MDX12), and the primary human fibroblasts (EDX1) were
each growth arrested using the following method. Fibroblasts were grown
in DMEM+10% FCS, 25 mM Hepes, 1 mM pyruvate, 2 mM L-Gln, 100 U/ml
penicillin, 100 μg/ml streptomycin in T75 flasks. At confluency, the
cells were detached and plated at a density of 105 cells/cm2,
further incubated for 48 h, then treated with mitomycin C (MMC) at 0, 2,
4, 8, 12 μg/ml for 5 h. The cells were then rinsed with PBS and
detached with 0.05% trypsin/0.02% EDTA. The remaining cells were plated
at densities of 100 to 5000 cells/cm2 in T25-flasks respectively (10
flasks for each density). These cells were incubated at 37° C.
with 2 media changes per week.

[0155] Efficiency of cell growth arrest was measured by weekly counting of
cells (using a hemacytometer) cultured in the flasks plated at a density
of 5000 cells/cm2 and by inspection of appearing colonies in the
flasks plated at a density of 100 cells/cm2. Changes in cell
morphology were also examined. A concentration of 8 μg/ml MMC was
sufficient to growth arrest the human primary fibroblasts (EDX1) and the
hTERT immortalized human fibroblast line (MDX12) agreeing with previous
data (Limat et al., J. Invest. Dermatol. 92:758-62 (1989)). The rat
fibroblast line (CRL 1213) showed a toxicity to MMC-concentrations above
4 μg/ml, while 2 μg/ml MMC proved to be optimal to growth arrest
these cells. The rat fibroblast cell line (1175/CRL 1213) transfected
with the FGF1-gene, was growth arrested at a concentration of 1 μg/ml
MMC. Concentrations below 1 μg/ml were not efficacious and higher
concentrations of MMC were progressively toxic. Mitomycin C-treated
fibroblasts (with the appropriate mitomycin-C dose), recovered from
cryogenic storage by thawing, displayed a cell recovery of at least 50%
(in agreement with Limat et al., In Vitro Cell Dev. Biol. 26:709-12
(1990)).

Example 4

Testing Effective Dose of Gamma Irradiation on Fibroblasts and
Keratinocytes

[0156] To measure the effect of gamma (γ) irradiation on the mitotic
activity of treated fibroblasts, a BrdU cell proliferation assay was
employed which functions in a 96 well format (Oncogene Research
Products). Non-immortalized, human primary fibroblast cells were treated
with γ irradiation at various doses, including 0, 60, 70, and 80
Grays. Cells were then plated at a density of 5,000 cells per well in a
96 well dish. Irradiated and non-irradiated controls were maintained in
culture for periods lasting 15 and 30 days. At each of these time points,
the mitotic activity was measured using a BrdU incorporation assay in
which BrdU incorporation in the DNA is assayed immunochemically and
measured via its absorbance at 450 nm.

[0157] In FIG. 1, non-irradiated cells treated with BrdU served as a
positive control for each experiment. Non-irradiated cells that were not
treated with BrdU served as the background absorbance, which was
subtracted from the absorbance at 450 nm for each sample (0, 60, 70, and
80 Grays) and the final data was normalized by setting the positive
control to 100% relative BrdU incorporation. In FIG. 1, it is clear that
treatment with γ irradiation at levels of 60 Grays and above
induces primary fibroblasts into a post mitotic state. A total of 3
samples was considered for each condition. Data is shown as the
average±SEM.

[0158] In FIG. 2, viability of cells following gamma (γ) irradiation
treatment was assessed by determining the adhesion of cells to normal
cell culture surfaces. Cells were plated in 6 well culture dishes at a
concentration of 9,500 cells/cm2 or 95,000 cells per well. Four
hours after plating cells, media was replaced with a fresh media
containing 50 μg/ml of Neutral Red and cells were incubated for 2
hours at 37° C. and 5% CO2. Wells were then rinsed twice with
NaCl 0.9% and dried overnight. The following day, the dye was dissolved
using a mix of 1:1 acidic acid (2%) and ethanol (95%). Each well was
incubated in 1 ml of this solution at room temperature for 15 minutes
after which 200 ul of the mix was measured for its dye content at 540 nm.
No significant difference was observed between the adherence of treated
versus non-treated cells indicating that gamma (γ) irradiation does
not effect cell viability and that cells may attached to a culture
surface following gamma (γ) irradiation. Each data point is the
average measurement of three separate 6 wells. Data is displayed as the
average±SD.

[0159] Gamma (γ) irradiation at 80 Gy was also evaluated for its
ability to induce differentiation of primary human keratinocytes into a
post mitotic state. Irradiated keratinocytes were plated on a layer of
growth arrested fibroblasts feeder cells (gamma (γ) irradiated at
70 Gy). Feeder cells were plated at a density of 5,000 cells/cm2 and
keratinocytes 12,500 cells/cm2. Keratinocyte growth and phenotype
were followed for 3 weeks thereafter by observation using an inverted
microscope. During this period, keratinocytes were observed to adopt a
differentiated phenotype, with cells increasing their size and area of
attachment. Keratinocytes cultured in this manner were not able to divide
and cover the culture surface, but instead remained either isolated or in
small clusters, indicating that irradiation had induced cells into a post
mitotic state.

Example 5

Testing Cell Densities with Fibrin Paste: Secretion of Growth Factors and
Cytokines by Mixtures of Keratinocytes and Fibroblasts in a Fibrin Matrix

[0160] Human primary keratinocytes and fibroblasts were growth arrested by
gamma (γ) irradiation at 80Gy prior to formulation. Fresh
preparations of human primary keratinocytes and fibroblasts were mixed at
a ratio of 1:9 at final concentrations including 2.5, 5, 10, 20 and 40
million cells/ml in a suspension containing 10% thrombin (Tisseel,
Baxter)+15% glycerol+5% Human serum albumin. In a second vial, a 25%
fibrinogen (Tisseel, Baxter) solution was prepared. A cell and fibrin
"paste" was prepared in individual wells of a 24 well plate by spraying
together I spray (130 μl) of the cell+thrombin suspension with 1 spray
(130 μl) of the fibrinogen suspension. Secretion of various growth
factors and cytokines by these cells into media was measured during day 2
following preparation of the cells in the fibrin matrix. Data shown in
Table 5 represents an average of 5 individual data points (samples) for
each condition presented.

[0161] Table 5 illustrates the variety of different growth factors are
actively secreted from keratinocytes and fibroblasts contained in a
fibrin matrix. It is also known in the art that bFGF produced by these
cells can be found in the fibrin matrix. The absolute levels of growth
factors produced were observed to be dependant on the particular nature
of the growth factor in question. Because biological potency and
half-life is molecule dependant, actual pg levels of independent growth
factors is not the primary interest. Rather, it is believed that the
biological action of the cocktail of molecules secreted from these cells
together offers a unique way of targeting many biological pathways
simultaneously. It is worth noting that physiological quantities of
growth factors and cytokines are being produced.

[0162] In Table 5, the secretion of the 5 different molecules appears to
be dose-dependant according to the cell concentration employed. This
holds true as cell concentration increases from 2.5 to 10 million
cells/ml. For most factors, except KGF and HGF, even higher secretion
levels are witnessed at 20 million cells/ml. However, once the cell
number increases to 40 million cell/ml, a drop in protein production is
observed for all molecules except IL-1 beta. This suggests that an
optimum cell concentration likely exists and that, as shown, each cell
concentration will lead to the production of a different protein profile
as seen in the Table 6 below. Table 6 was generated by normalizing the
secretion of VEGF, KGF, HGF and IL-1 beta to that of GM-CSF. This
illustrates that, for different cell concentrations, there exist a
different protein profile, with molecules being produced at different
ratios depending on the cell concentration under consideration.

[0163] The allogeneic cell-based treatment (named Allox) under
investigation is a two component product which can be applied topically
to chronic ulcers using a spray applicator. Upon spraying the two
components on the wound site, a fibrin matrix is created that traps the
applied cells at the region of the ulcer, permitting local release of
trophic factors by these cells.

[0165] The objective of this study was to assess the effects Allox on the
wound healing of chronic ulcers, to assess its safety and tolerability in
a patient population, and to determine the effect of the product on the
incidence of complete wound closure of chronic leg ulcers in patients
with venous or arteriovenous insufficiency.

Methodology

[0166] Patients over 18 years of age with at least one venous or combined
arterio-venous ulcer between the ankle and the knee who met the
protocol's eligibility criteria were recruited to receive Allox in the
C2001 study. At study day (SD)-14, patients were found eligible and
recruited to receive the Allox treatment. At SD1, patients received the
first application of the study treatment, which was repeated on a weekly
basis up to 8 weeks, or until ulcer closure, whichever came first. A
follow-up visit occurred 4 weeks following the last application of the
treatment.

Patient Number

[0167] A total of 13 patients were initially enrolled, one of whom was
determined to be not eligible for the protocol at a later time.

Diagnosis and Main Criteria for Inclusion

[0168] Patients over 18 years of age with one or more venous or combined
arterio-venous ulcers between the ankle and the knee whose etiology was
confirmed by Ankle Brachial Pressure Index, by Ankle and/or Great Toe
pressure, and by Duplex Ultrasound were eligible for this study. At SD-14
patients had to have an ulcer longer than 1 month duration with a surface
area greater than 1 cm2. Patients were required to be capable of
communicating and cooperating with the Investigator and other staff and
had to provide informed written consent. Female patients must have been
post-menopausal or surgically sterilized.

Test Treatment and Mode of Administration

[0169] Application by sequential spraying of the solutions provided in the
two bottle kit. A total 0.4 ml of product was applied alternatively: 2
sprays of component #1 (fibrinogen) followed by 2 sprays of component #2
(cells+thrombin). This procedure was repeated twice. Such an application
covers 12 cm2 of ulcer area, and each cm2 of treated wound
received a total of 0.25×106 cells.

Duration of Treatment

[0170] At SD1, patients received the first application, which was repeated
on a weekly basis up to 8 weeks, or until ulcer closure, whichever came
first. A follow-up visit would occur 4 weeks following the last
application. The study duration period was 14 weeks, consisting of a
2-week run-in period, an 8 week treatment and a follow up period of 4
weeks.

Criteria for Evaluation

[0171] Efficacy was assessed by noting complete closure of the ulcer;
ulcer surface area at W8 and W12 compared to SD1; edge effect; and ulcer
symptoms.

[0172] Safety was monitored by following the frequency and severity of
adverse events until week 12. Standard laboratory tests, physical
examinations and vital sign measurements were also recorded.

Statistical Methods

[0173] Given the small number of patients treated, no statistical analysis
was performed. Rather, results are presented herein in descriptive form.

[0175] A total closure was observed for one ulcer at SD57 and for two
ulcers at the week 4 follow-up. For 5 patients, an improvement defined as
more than 30% decrease in surface area was noted. Treatment failure,
which was defined as either no reduction in ulcer size or an increase of
ulcer size during the study period, was observed in 4 patients treated
with Allox.

Clinical Study Conclusions

[0176] Weekly Allox treatments over an 8 week period were determined to be
safe and non-toxic to patients with chronic leg ulcers. The two ulcer
infections that were potentially treatment related resolved before the
study end. Mild ulcer infections are relatively common place if correct
hygiene is not respected.

[0177] Five patients showed greater than 30% reduction of area at the end
of the follow up period, with 2 patients displaying complete ulcer
closure. From this small study, a tendency towards response was observed
in "younger" ulcers (<6 months).

[0178] Fibrin is one potential biological polymer that can be employed for
suspending and trapping cell mixtures for therapeutic purposes.
Polymerized fibrin is created by mixing fibrinogen and thrombin together
at appropriate concentrations. In the matrix shown in FIG. 3, various
dilutions of the fibrinogen(Tisseel, Baxter) and thrombin(Tisseel,
Baxter) were tested for their effect on the polymerization process and
generation of the end product fibrin. Dilutions considered ranged from
1/4 to 1/80 of the original fibrinogen and thrombin components supplied
in the TissuCol kit. In the original Tisseel kit, fibrinogen had a
concentration of 75 to 115 mg/ml, while thrombin had a concentration of
500 IU/ml. For this study, the fibrinogen was diluted in HBSS without
Ca2+ and Mg2+ and the thrombin was diluted in HBSS with
Ca2+ and Mg2+.

[0179] Fibrin characteristics considered included polymerization time
(seconds), consistency, and mechanical strength. The fibrin polymer was
generated using a spray technique by which one spray of fibrin was
combined with one spray of thrombin in a single well of a 24 well culture
plate. Conditions were repeated in triplicate. The spray volume employed
was 130 μl per spray.

[0180] All dilutions considered permitted the formation of a fibrin
polymer, though the properties of the fibrin polymer varied widely
depending on the dilutions employed. The consistency and the mechanical
strength of the fibrin were rated using the scale (-, +, ++, +++) in
witch - was considered poor and +++ was considered excellent. Ratings of
++ or +++ were considered to be acceptable for potential use to suspend
cells for therapeutic applications. The conditions for the maximal
dilution of fibrinogen was 1/20 fibrinogen and 1/8 thrombin, while the
conditions for the maximal dilution of thrombin was 1/4 fibrinogen and
1/40 thrombin.

[0181] Additionally, normal plasma can also be used as a matrix material
for the production of a biological glue to trap cells at the application
site of a wound. Mixtures made by pipetting normal undiluted human plasma
together with thrombin at a dilution of 1/50 permitted the formation of a
fibrin clot or polymer. This fibrin polymer showed handling
characteristics similar to the commercially available fibrin glues,
suggesting that it can serve as a substitute to Baxter's Tisseel VH or
Tissucol and Haemacure's APR concentrated fibrin-based products.

[0182]FIG. 4 shows the secretion of growth factors from cells entrapped
in a fibrin matrix, according to the methods of the instant invention.
VEGF and GM-CSF secretion was assessed from medium conditioned for 24
hours during the second day following production of the fibrin cell
matrix. bFGF secretion was measured in extracts obtained from the cell
matrix 48 hours after production. To growth arrest cells, both primary
human fibroblasts and keratinocytes were treated with 8 ug/ml mitomycin
during five hours. Cells were rinsed with HBSS without Ca2+ and
Mg2+ prior to trypsinization.

[0183] Cells were applied using a spray pump delivering 50 μl per
spray.

[0188]FIG. 4 compares the quantity of secreted growth factors produced by
cell and fibrin preparations following spray of different cell doses into
individual wells of a 24 well culture dish. The figure also shows growth
factor secretion quantities when cell and fibrin preparations are made by
simple pipetting (non-sprayed). A comparison of sprayed versus
non-sprayed preparations indicates that there is a decrease in measured
VEGF secretion, while GM-CSF and bFGF secretion levels are comparable.
Additionally, an increase from 2 to 4 sprays led to higher secretion
levels of growth factors by the cells, which highlights the possibility
of dosing growth factors by altering the number of cells. Secreted GM-CSF
and VEGF were dosed in the culture media, while bFGF was dosed in the
fibrin matrix. Data is presented as the average+SEM (n=4, spray; n=3,
non-sprayed).

Example 9

Comparison of Growth Factor Production by Different
Keratinocyte:Fibroblast Cell Ratios

[0189] In FIG. 5A and FIG. 5B, a comparison is made of growth factors
released from cells entrapped in a fibrin matrix. The fibrin cell matrix
is formed by spraying either one or two sprays of component #2
(cells+thrombin) with one or two sprays of component #1 (fibrinogen). A
concentration of 15×106 cells/ml in component #2 was employed
and the spray pump used delivered a volume of 70 μl per spray. To
growth arrest cells, both primary human fibroblasts and keratinocytes
were treated with 8 ug/ml mitomycin during 5 hours. Cells were rinsed
with HBSS without Ca2+ and Mg2+ prior to trypsinization.

[0191] FIGS. 5a and 5b demonstrate that mixing keratinocytes and
fibroblasts at different ratios, while maintaining a constant total
number of cells, gives rise to variable growth factor secretion
characteristics. As keratinocytes are added to the fibroblasts, an
increase in GM-CSF secretion is observed. For one spray, at
keratinocytes:fibroblasts ratios of 1:24 to 1:8 a plateau is reached for
GM-CSF production. Further addition of keratinocytes to the second
component of the cell preparation of the invention does not appear to
provide an advantage in terms of GM-CSF secretion.

[0192] Considering two spray preparations, at a keratinocytes:fibroblasts
ratios of 1:49 to 1:1 the GM-CSF secretion passes 5000 pg/day. While
GM-CSF secretion is highest at a keratinocyte:fibroblast ratio of 1:4, it
is apparent that the largest increase in GM-CSF secretion is gained when
passing from a ration of 1:99 to 1:49.

[0193] Moreover, it is also evident that VEGF production is highly
dependant on the ratio of keratinocytes to fibroblasts in component #2.
In the experiments detailed in FIGS. 5a and 5b, there appears to be
optimal secretion of VEGF near a keratinocytes:fibroblasts ratio of 1:8,
since further increasing the number of keratinocytes leads to a decrease
in the quantity of VEGF secreted.

[0194] At different cell ratios, the application of a greater number of
sprays also leads to an increase in growth factor secretion levels. Data
is shown as average±SEM (n=4, 1 spray; n=3, 2 sprays).

Example 10

Comparison of Storage at -160° C. Versus -80° C. During One
Week

[0195]FIG. 7 shows a comparison of growth factor secretion by cells
stored cryopreserved at -160° C. versus at -80° C. for a
period of one week. The cryoprotectant used in this experiment was a 10%
glycerol solution with 10% thrombin (Tisseel, Baxter) and the
keratinocyte:fibroblast ratio employed was 1:1. Prior to
cryopreservation, cells were detached from their culture surfaces using
trypsin and subsequently irradiated using gamma (γ) irradiation at
80Gy. A controlled rate freezer was used to gradually cool cell
preparations to -80° C. After thawing one week later, one spray
(1300) of the cell preparation (1.3 million cells at 10 million cells/ml)
were spray mixed with one spray (130 μl) of fibrinogen in single wells
of a 24 well petri dish. The results show GM-CSF, VEGF, and bFGF
secretion during day 2 for three samples stored for one week at
-160° C. and four samples stored for one week at -80° C.
compared to a control fresh (unfrozen) sample containing the same
cryoprotectant. Secreted GM-CSF and VEGF were dosed in the culture media,
while bFGF was dosed in the fibrin matrix. Secretion data indicates that
both -80° C. and -160° C. are suitable for storage, though
-160° C. may be preferable when using a 10% glycerol solution.
Data is shown as average±SEM (n=4).

Example 11

Comparison of Secretion After One Week Storage at -80° C. in 10%
Glycerol Versus 15% Glycerol

[0196]FIG. 8 shows a comparison of GM-CSF, VEGF and bFGF secretion by
cells cryopreserved at -80° C. in 10% glycerol versus in 15%
glycerol for a period of one week. The keratinocyte:fibroblast ratio used
in this example was 1:1 mixed with 10% thrombin (Tisseel, Baxter) and a
cryoprotectant. Prior to cryopreservation, cells were detached from their
culture surfaces using trypsin and subsequently irradiated using gamma
(γ) irradiation at 80Gy. A controlled rate freezer was used to
gradually cool cell preparations to -80° C. After thawing one week
later, one spray (130 μl) of the cell preparation (1.3 million cells
at 10 million cells/ml) were spray mixed with one spray (130 μl) of
fibrinogen in single wells of a 24 well petri dish.

[0197] The results show GM-CSF, VEGF, and bFGF secretion during day 2 for
four samples stored for one week at -80° C. using 10% glycerol and
four samples stored for one week at -80° C. using 15% glycerol
compared to control fresh (unfrozen) samples containing the same
cryoprotectant concentrations. A trend of higher protein secretion was
observed in samples stored in 15% glycerol versus those kept in 10%
glycerol. Secreted GM-CSF and VEGF were dosed in the culture media while
bFGF was dosed in the fibrin matrix. Data is presented as average±SEM
(n=4).

[0198]FIG. 9 shows a comparison of growth factor secretion by cells
cryopreserved at -80° C. in 15% glycerol versus 15% glycerol+5%
Human Serum Albumin (HSA) (Griffols) for a period of one week. The
keratinocyte:fibroblast ratio used in this example was 1:3 mixed with 10%
thrombin (Tisseel, Baxter) and a cryoprotectant. Prior to
cryopreservation, cells were detached from their culture surfaces using
trypsin and subsequently irradiated using gamma (γ) irradiation at
80Gy. A controlled rate freezer was used to gradually cool cell
preparations to -80° C. After thawing one week later, one spray
(130 μl) of the cell preparation (1.3 million cells at 10 million
cells/ml) were spray mixed with one spray (130 μl) of fibrinogen in
single wells of a 24 well petri dish. The results show GM-CSF, VEGF, and
bFGF secretion during day 2 for three samples stored for one week at
-80° C. using 15% glycerol and three samples stored for one week
at -80° C. using 15% glycerol+5% HSA (Griffols) compared to
control fresh (unfrozen) samples containing the same cryoprotectants.
Secretion data indicates that the addition of Human Serum Albumin
improves the frozen product formulation by permitting higher protein
secretion levels. Data is presented as average±SEM (n=4).

Example 13

Bioactivity of Keratinocyte and Fibroblast Mixtures Following Long-Term
Storage at -80° C.

[0199]FIG. 10 details the secretion of the human proteins GM-CSF and VEGF
by cell preparations following storage at a temperature of -80° C.
for extended periods. Data from three separate clinical production
batches is shown. Batches containing a ratio of human primary fibroblasts
to keratinocytes of 1:1 at a final concentration of 10×106
cells/ml were irradiated using gamma (γ) irradiation at 80Gy and
frozen in a solution containing thrombin, 15% glycerol and 5% human serum
albumin. Samples from each production batch were thawed following 1, 4,
8, and 12 weeks storage at -80° C. Thawed samples were
subsequently sprayed into 24-well plates for testing. In individual
wells, a single spray (130 μl) of cells+thrombin+cryoprotectant is
mixed with a single spray (130 μl) of fibrinogen. The mixture of these
two sprays creates a fibrin polymer matrix containing living fibroblasts
and keratinocytes. The secretion of proteins by cells trapped in the
fibrin matrix is measured during day 2 (the period lasting from 24 hours
to 48 hours after thawing).

[0200] Secretion of GM-CSF and VEGF into the media by thawed cell
preparations remains relatively stable over a period of storage lasting 3
months at -80° C. Slight variations in GM-CSF secretion was seen
from individual batch to batch, though secretion within a batch appeared
to be relatively stable. Secreted VEGF appeared stable, on average, both
from a batch to batch perspective as well as within individual production
batches. This data reveals that, with respect to GM-CSF and VEGF
secretion, the product is stable in a cryogenic state at -80° C.
for periods lasting at least 12 weeks. Data is presented as
average±SEM.

Example 14

Comparison of Secretion After One Week Storage at -80° C. for
Different Keratinocyte:Fibroblast Ratios

[0201]FIG. 11 shows the secretion of growth factors from cryopreserved
cell preparation formulations following one week of storage at
-80° C. The graph shows differences in secretion for various human
primary keratinocyte:fibroblast ratios, including 1:0, 1:1, and 1:9 as
well as differences associated with total cell concentrations of 5, 10
and 20 million cells/ml. Prior to cryopreservation, cells were detached
from their culture surfaces using trypsin and subsequently irradiated
using gamma (γ) irradiation at 80Gy. A controlled rate freezer was
used to gradually cool cell preparations to -80° C. After thawing
one week later, one spray (130 μl) of the cell preparation (5, 10 and
20 million cells/ml) were spray mixed with one spray (130 μl) of
fibrinogen in single wells of a 24 well petri dish.

[0202] A comparison of the secretion data for the 1:0 ratio and the 1:1
ratio showed the importance of adding fibroblasts to the keratinocytes in
terms of GM-CSF and VEGF production. In all cases, there was a cell-dose
dependant relationship with the secretion of the growth factors during
the second day after thawing. The data also demonstrated that reducing
the number of keratinocytes (i.e. by reducing the keratinocyte:fibroblast
ratio to 1:9) did not lead to a reduction in overall growth factor
secretion. These results were in accordance with the data obtained for
"fresh" (non-frozen) cell preparations (see Example 9, supra) and
suggested that only minimal quantities of keratinocytes were needed to
produce the synergistic effect achieved by mixing the keratinocytes and
fibroblasts together. This study also demonstrated that fibroblasts play
an important role in the production of VEGF by cell preparations.

[0203] Prior to cryopreservation, cells were detached from their culture
surfaces using trypsin and subsequently irradiated using gamma (γ)
irradiation at 80Gy. Cell concentrations tested included 5, 10, and 20
million cells/ml (with a keratinocyte:fibroblast ratio 1:1), each mixed
with 10% thrombin (Tisseel, Baxter) and the cryoprotectant. A controlled
rate freezer was used to gradually cool cell preparations to -80°
C. After thawing one week later, one spray (130 μl) of the cell
preparation (5, 10 and 20 million cells/ml) were spray mixed with one
spray (130 μl) of fibrinogen in single wells of a 24 well petri dish.
To assess variability in frozen (cryopreserved) samples, 3 separate tubes
for each condition were cryopreserved for one week at -80° C. Upon
thawing, five samples were made per tube. The data presented in Table 8
is the average of the 15 samples available for each condition. The
reproducible secretion data observed in the three frozen and thawed tubes
per condition attests to the quality of the cryopreservation method. For
fresh preparations, either 3 or 4 samples were made. Secretion of GM-CSF
and VEGF by both freshly trypsinized and frozen cell preparations was
observed to increase as cell density in the fibrin matrix increased from
2.5 to 5 to 10 million cells/ml (corresponding to 5, 10 and 20 million
cells/ml found in original frozen preparations). This further illustrates
the potential of dosing therapeutic effects by cell-based treatments.

Evaluation of the Safety of the Frozen Cell Preparation of the Invention

[0204] The safety of the frozen wound-healing cell preparation of the
invention has been evaluated in a multicenter, open phase I study. The
cell preparation consisted of a kit containing two components. Component
#1 was a suspension of fibrinogen and component #2 was a suspension of
keratinocytes and fibroblasts mixed in thrombin and cryoprotectant. Cell
preparations were frozen at -80° C., shipped to the clinic site at
-80° C. and stored at -80° C. on site until use.
Immediately prior to use, the cell preparation was thawed in a heated
water bath. After thawing the two components (fibrinogen) and
(cell+thrombin+cryoprotectants) were sequentially spray applied on the
wound site, forming a thin fibrin matrix containing living keratinocytes
and fibroblasts. Fourteen patients with chronic venous leg ulcers not
responding to standard treatment with dressings and compression for at
least 4-weeks (run-in phase) were enrolled in 5 centers in the
Netherlands and Dutch Antilles. Ulcer sizes at baseline ranged from 0.3
to 20.4 cm2 (mean 5.8). Concomitant to the standard treatment, the cell
preparation was then applied once weekly for up to 12 weeks or until
complete closure, whichever came first.

[0205] No serious adverse events were reported in relation to this cell
preparation. Four moderate to severe adverse events were thought to be
attributable to the cell preparation (3×ulcer pain, 1× with
increasing ulcer size). Moreover, there were no clinical signs of wound
infection. Complete closure at week 12 was observed in 10 patients, 7
within 4 weeks and 3 within 4 to 12 weeks of treatment. Mean time to
closure was 5.4 weeks.

[0206] In conclusion, the cell preparation of the invention is safe and
well tolerated for the treatment of chronic venous leg ulcers.

Example 17

Spray Applied Living Keratinocytes and Fibroblasts as a Biologically
Active Wound Dressing

[0207] Primary fibroblasts and keratinocytes residing in the skin,
naturally secrete a cocktail of growth factors and cytokines that act to
stimulate the wound healing response following interruption of the
cutaneous barrier. To mimic this natural process, a living cell-based
wound dressing was developed for the treatment of chronic venous ulcers.
It consists of two components: 1) a solution of fibrinogen; and 2) a
suspension of keratinocytes and fibroblasts in thrombin and
cryoprotectant. The product is stored frozen at -80° C. until use,
at which time it is thawed, with the two components applied sequentially
to the wound surface using a spray applicator. In this manner,
polymerization of the fibrin occurs on the wound with delivered cells
becoming trapped in a thin layer of fibrin at the ulcer site.

[0208] Mixtures of allogeneic, growth-arrested primary keratinocytes and
fibroblasts were observed to secrete different levels of therapeutic
proteins (VEGF, HGF, GM-CSF, bFGF, and KGF) depending on the ratios
employed. Secretion of GM-CSF was dependant on the synergy derived from
the mutual presence of fibroblasts and keratinocytes. Increasing the cell
concentration in the final wound dressing from 1.25×106
cells/ml to 5×106 cells/ml led to an elevated secretion of
growth factors and cytokines. Preliminary studies have shown that cell
preparations remain biologically active for at least 2 months when stored
at -80° C.

Other Embodiments

[0209] It is to be understood that while the invention has been described
in conjunction with the detailed description thereof, the foregoing
description is intended to illustrate and not limit the scope of the
invention, which is defined by the scope of the appended claims. Other
aspects, advantages, and modifications are within the scope of the
following claims.